The major aim of this project is to elucidate mechanisms controlling cell fate decisions in developing T cells. Precursor T cells undergo a testing process in the thymus to ensure that cells expressing useless or self-reactive T cell antigen receptors (TCR) do not mature. These selection processes require TCR engagement with MHC ligands expressed by thymic stromal cells, but the precise nature of these interactions determines whether the cells will survive, mature, or die. Thymocytes also receive signals as they mature that direct them into specific lineages. Expression and signaling through the gamma-delta TCR or the pre-TCR impose a bias on lineage choice that determines whether early T cell precursors specify the gamma-delta or alpha-beta T cell fate. Immature thymocytes that choose the alpha-beta pathway must express a mature alpha-beta TCR appropriate for positive selection and continued maturation. Quantitative and qualitative aspects of TCR signaling determine if the thymocyte will be positively or negatively selected and whether it will mature in the CD4 or CD8 T cell lineage. In addition, signals through the TCR must integrate with other developmental signals, such as those regulated by Notch, to guide precursors to the appropriate fate. In efforts to understand how Notch signaling is regulated in the thymus, we have generated mutant mice for assessing Notch function in vivo that circumvent problems of redundancy in Notch receptors/ ligands, early lethality associated with deletion mutants, and those associated with ectopic and over-expression. In one approach, we generated transgenic mice expressing a truncated form of Presenilin, the protein responsible for generating the active form of Notch. This mutant operates as a dominant negative in thymocytes, resulting in a profound block in both gamma-delta and alpha-beta T cell development while enhancing production of B and NK cells. Moreover, in the highest expressing transgenic line, because of the severe developmental block, we have identified a population of thymocytes that cannot be detected in the normal thymus. We believe this cell type could be the long sought thymus seeding progenitor. Most important, co-expression of an activated form of Notch (gain-of-function mutant) corrects the developmental defects imposed by Presenilin mutations, indicating that the abnormal phenotypes are due to interference with Notch signaling. Also to interfere with Notch signaling, we have generated conditional null mutations of Presenilin1/2 (PS) that allow stage- and tissue-specific expression. In one model that conditionally deletes Presenilin genes globally, we observe all the phenotypic defects in epithelial and lymphoid development previously reported to be associated with Notch signaling mutants, as well as an unreported defect in dendritic cell maturation. In another model, the generation of mature CD4 T cells is attenuated. Precursor thymocytes express normal levels of TCR and the activation marker, CD69, but the level of CD5 (a negative regulator of TCR signaling) is dramatically reduced. These findings raise the possibility that diminished TCR signaling during positive selection is responsible for the decreased generation of CD4 T cells in PS-deficient thymocytes. Indeed, PS-deficient thymocytes flux calcium poorly in response to TCR stimulation in vitro. Moreover, positive selection of PS-deficient thymocytes is improved by introduction of a higher affinity MHC ligand, demonstrating that diminished TCR signaling in the absence of PS can be compensated for by increased TCR/MHC affinity. These findings raise the issue of whether Notch exerts its effects on TCR signaling by directly interacting with the TCR or components of TCR signal transduction, or through changes in gene expression that are regulated by Notch signaling One of many Notch regulators is the adapter protein, Numb, which is known to antagonize Notch signals in Drosophila neural development. Of relevance, Numb contains several signaling motifs; a phosphotyrosine binding domain and a proline-rich region containing putative SH3-binding domains. A recent study shows that Numb co-localizes with TCR and Notch in T cells and Numb can be co-precipitated with Lck, c-Cbl, and Vav in thymocytes. Numb is reported to regulate receptor-mediated endocytosis and to be involved in the ubiquitination and degradation of Notch and other transmembrane receptors. Because of its potential to interact with Notch and the antigen receptor signaling pathways, we targeted the expression of a transgene encoding a dominant negative form of Numb (dnNumb) to developing lymphocytes. This truncated form of Numb, consisting only of the PTB domain, has the potential to inhibit the function of Numb and its homologue, Numb-like, that are both expressed in mouse thymus. Interfering with Numb activity reduces lymphocyte numbers in the periphery, with the greatest effects on B cells and CD8 T cells. The reduction in mature lymphocyte numbers can be largely attributed to defects in proliferation and differentiation that are associated with pre-TCR and pre-BCR signaling in early lymphocyte development. Interestingly, the efficiency of generating CD4 T cells is improved in dnNumb mice with a diverse TCR repertoire. However, no CD4 T cells arise in dnNumb mice with a fixed MHC class II-specific TCR, which is perhaps a consequence of negative selection. Although the dnNumb was expected to act as a Notch gain-of-function mutant, the phenotypic changes observed have little similarity to those reported for mutant mice expressing an activated form of Notch. Our preliminary results suggest instead that Numb may be involved in the internalization and, or degradation of the TCR. If this is the case, the effect of dnNumb may be to increase TCR surface expression, thereby enhancing TCR signaling. It is noteworthy that Numb functions as an adapter by binding conserved NPXY sequence motifs. Interestingly, this motif exists in the CD3e (ITAM), a component of the TCR complex that mediates signal transduction. Collectively, these studies indicate that both TCR and Notch influence cell fate decisions at several developmental stages and provide some hints of how these two signaling pathways may be associated. In other efforts to understand how TCR signals promote cell fate decisions, we are interested in how the pre-TCR promotes the development and commitment of early thymocyte precursors to the alpha-beta T cell lineage. The pre-TCR is comprised of a newly rearranged TCRbeta chain and an invariant pre-TCRalpha chain. Signals from the pre-TCR lead to allelic exclusion at the TCRbeta locus, proliferation, and differentiation. The pre-TCRalpha chain is a transmembrane protein consisting of an extracellular domain, and an intracellular 30 amino acid proline rich tail. Although the proline rich tail is unique both for its length and intrinsic signaling motifs, the contribution of the cytoplasmic tail to pre-TCR signaling and function has not been defined. To study the signaling potential of the pre-TCRalpha tail under normal physiological regulation, we used gene targeting and homologous recombination to induce a truncation of the pre-TCRalpha locus, resulting in a mutant pre-TCRalpha protein lacking the intracellular tail. In these tailless pre-TCRalpha mice, there is a partial block in development, resulting in fewer thymocytes and a perturbation the cell cycle kinetics associated with pre-TCR signaling. However, when progenitors of the mutant are placed in direct competition with wild type progenitors in hematopoietic chimaeras, wild type cells are nine times more efficient at reconstituting the irradiated thymus. These results support a role for the cytoplasmic tail in the pre-TCR-dependent cell cycle progression required for the efficient proliferation of early thymocytes.